Characterization and functional analysis of cathelicidin-MH, a novel frog-derived peptide with anti-septicemic properties

Abstract

Antimicrobial peptides form part of the innate immune response and play a vital role in host defense against pathogens. Here we report a new antimicrobial peptide belonging to the cathelicidin family, cathelicidin-MH (cath-MH), from the skin of Microhyla heymonsivogt frog. Cath-MH has a single α-helical structure in membrane-mimetic environments and is antimicrobial against fungi and bacteria, especially Gram-negative bacteria. In contrast to other cathelicidins, cath-MH suppresses coagulation by affecting the enzymatic activities of tissue plasminogen activator, plasmin, β-tryptase, elastase, thrombin, and chymase. Cath-MH protects against lipopolysaccharide (LPS)- and cecal ligation and puncture-induced sepsis, effectively ameliorating multiorgan pathology and inflammatory cytokine through its antimicrobial, LPS-neutralizing, coagulation suppressing effects as well as suppression of MAPK signaling. Taken together, these data suggest that cath-MH is an attractive candidate therapeutic agent for the treatment of septic shock.

FM-CATH, A Novel Cathelicidin From Fejervarya Multistriata, Shows Therapeutic Potential for Treatment of CLP-Induced Sepsis

“…Both sexes of adult F. multistriata (n 3; weighing about 40 g) with no specific permissions in need for sampling were captured from Guangzhou city, Guangdong Province, China (23.12°N, 113.28°E) and were euthanized by CO 2 before their skins were subsequently sheared and preserved in liquid nitrogen. Total RNA of the F. multistriata frog skin was extracted with Trizol (Life Technologies, CA, United States) according to the manufacturer's protocols and prepared as temples for PCR amplifications as previously reported by us (Chai et al, 2021). The sense primer (5′-GGATGAAGGTCTGGCAGTGTGTGC-3′) was used for the 5′ sequence PCR amplifications of cDNA as described in our previous study (Chai et al, 2021).…”

“…Total RNA of the F. multistriata frog skin was extracted with Trizol (Life Technologies, CA, United States) according to the manufacturer's protocols and prepared as temples for PCR amplifications as previously reported by us (Chai et al, 2021). The sense primer (5′-GGATGAAGGTCTGGCAGTGTGTGC-3′) was used for the 5′ sequence PCR amplifications of cDNA as described in our previous study (Chai et al, 2021). Prediction of physical and chemical parameters of FM-CATH was done by the ExPASy-ProtParam tool (http://web.expasy.org/protparam/).…”

“…Cells were grown in DMEM or RPMI 1640 medium, or medium containing continuous concentrations of FM-CATH (2.5, 5, 10, 20, 40 μM) at 37°C for 24 h before MTT was added in the dark and the culture was continued for 4 h. The supernatant was removed and DMSO was added before the absorbance at 490 nm was measured. Hemolytic assay was carried out with fresh blood from mouse heart as reported in our previous article with minor modification (Chai et al, 2021). In short, 2% of mouse erythrocyte suspension in TBS solution (v/ v) was treated with 2.5, 5, 10, 20, and 40 μM FM-CATH in a 96-V-well plate at room temperature for 2 h. 1% Triton X-100 and PBS were applied as the positive and negative control, respectively.…”

“…PlexArrayTM HT A100 system (Plexera LLC, Bothell, Washington, United States) and bare gold SPRi chip (Nanocapture gold chip, with a gold layer or 47.5 nm thickness) was used to explore the real-time binding reaction of FM-CATH with LPS, LTA, and proteases as described in our previous method (Chai et al, 2021). In detail, FM-CATH (2 mM) and BSA dissolved in PBS were spotted in multiplex onto the gold chip surface and then stored at 4°C for 14 h in a humid box according to the instruction of the manufacturer.…”

“…CLP was carried out as described by us with minor modifications (Chai et al, 2021). In brief, six-week-old BALB/c mice of either gender were randomly grouped to sham, CLP, and FM-CATHtreated groups (n 6).…”